Thrombosis, the formation and development of a blood clot or thrombus within the vascular system, while a life saving process when it occurs during a hemorrhage, can be life threatening when it occurs at any other time. The thrombus can block a vessel and stop blood supply to an organ or other body part. If detached, the thrombus can become an embolus and occlude a vessel distant from the original site. Thrombotic disorders now constitute one of the major causes of mortality in both developing and developed countries world-wide. Although still the most preferred emergency (“SOS”) medication against thrombotic circulatory disorders, the plasminogen activator protein drugs, such as streptokinase (SK), staphylokinase (SAK) and tissue plasminogen activator (tPA), are slowly losing their supremacy to emergency cardiac interventions such as stenting and bypass surgeries in the affluent countries because of bleeding risks associated with their use, and also the often-encountered problem of clot reformation at the same site of vascular injury due to thrombin activity and/or fresh generation of thrombin. Thus, there is an acute need to develop smarter and more effective thrombolytic drugs with additional features such as clot specificity and anti-thrombotic properties.
Blood clot formation is an end-result of a complex set of cascade reactions wherein several biochemical events cause the sealing, or repair, at the site of injury (Butenas and Mann 2002). On the basis of initiation of the blood coagulation cascade, pathways have been divided into extrinsic and intrinsic pathway, wherein the extrinsic pathway is initiated by the exposure of tissue factor (TF), whereas the intrinsic pathway is initiated by factor XII (Hageman factor), high molecular weight kininogen (HK), or prekallikrain. However, both cascade pathways ultimately join at the point of factor Xa generation, and subsequently follow common thrombin mediated fibrin generation (Cannon and Tracy 1995).
Thrombin generation is the central step of the blood coagulation process in vertebrates. During the thrombin generation process small amount of thrombin gets incorporated into the fibrin clot/network as it expands, and the catalytic site of this protease, being free in this absorbed thrombin, is able to amplify the clot growth (Liu, Nossel et al. 1979; Vali and Scheraga 1988). Thrombin interacts with diverse substrates and activates several clot promoting factors e.g. (a) it causes the activation of platelets by cleaving their cognate receptors (b) causes feed-back activation of factor V, VIII and XI (c) cause the activation of transglutminase, factor XIII and (d) converts fibrinogen into fibrin. Once the clot forms and stabilizes by inter-strand cross-linking, under the pathological conditions, it impedes the normal blood flow in vessels and leads to blockage of arteries and veins.
One of the most common, and preferred, medications against circulatory disorders emanating from pathological thrombus formation in animals/humans such as myocardial infarction, is the intravenous infusion of thrombolytic agents (Lijnen and Collen 1988; Collen and Lijnen 1990; Francis and Marder 1991). Available thrombolytics such as streptokinase (SK), urokinase (UK) and tissue type plasminogen activator (tPA) essentially operate through the same, plasmin-dependent mechanism (since these are plasminogen activators), where these cleave the scissile peptide between residues 561 and 562 of plasminogen and convert it into its proteolytically activated form, plasmin. Tissue type plasminogen activator and urokinase are proteases that specifically recognize the scissile peptide bond in human plasminogen (direct activators), whereas streptokinase and staphylokinase (which are protein ‘co-factors’ rather than proteases and thus ‘indirect’ activators; (see: (De Renzo, Boggiano et al. 1967; Buck, Hummel et al. 1968; McClintock and Bell 1971) first make tight 1:1 complexes with plasmin or plasminogen, and the resultant proteolytically active complex(es) cleave the scissile peptide bond of other, ‘free’ plasminogen molecules and exponentially generate plasmin (Reviewed by: (Wohl, Summaria et al. 1978; Castellino and Powell 1981; Wohl, Sinio et al. 1983; Davidson, Higgins et al. 1990). Of all the currently available clot-busters, that is, plasminogen activator protein drugs, streptokinase exhibits the highest thrombolytic power, although, being (like SAK) of bacterial origin, it has the limitation of engendering immune reactions in a small minority of patients. Nevertheless, it is widely used as an affordable thrombolytic because of its relatively low cost as compared to tPA and UK.
Successful thrombolytic therapy helps maintain normal blood flow and improves the survival in a significant number of patients (Verstraete 1990), but early re-occlusion or re-thrombosis, often at the same site, has continued to limit the successful application of thrombolytic drugs. Several studies demonstrate that early reocclusion occurs in up to 30% of patients after thrombolytic therapy (Ohman, Califf et al. 1990). The cause of rethrombosis or early reocclusion is explained by plasmin activity which increases the hyper coagulability of blood (Eisenberg, Miletich et al. 1988); it is proposed that plasmin activates contact factors (Ewald and Eisenberg 1995), factor V (Lee and Mann 1989) and likely also prothrombin (Seitz et al., 1993). Another suggested reason is the exposure of clot-bound thrombin subsequent to the latter's dissolution, which, in turn, generates more thrombin, with fibrin-bound thrombin being relatively resistant to anti-thrombin inhibitor/s (Hogg and Jackson 1989; Weitz, Hudoba et al. 1990); the ‘released’ thrombin again locally activates the procoagulant activity and starts to activate platelets (Kumar, Beguin et al. 1994; Puri, Kumar et al. 1995), thereby promoting a cycle of biochemical events leading to rethrombosis. Thus, inhibition of the thrombin at the site of injury both directly, and at the ‘secondary’ level of its procoagulant activity, should greatly thwart the above-described, unwanted chain of events. If such a property is integrated successfully in the same molecule as the thrombolytic drug, the advantages in terms of lives saved are obvious.
Plasminogen activators are a family of proteases which characteristically catalyse the enzymatic conversion of plasminogen to plasmin. TPA the enzymatic conversion of plasminogen to plasmin through the hydrolysis of a single Arginine-Valine bond.
Tissue plasminogen activator (also known as fibrinokinase, extrinsic plasminogen activator, t-PA or TPA) is a glycoprotein and has an approximate molecular weight (MW) of about 70,000 Daltons (68,000 Daltons). It is a serine protease which catalyses the enzymatic conversion of pro-enzyme plasminogen to active enzyme plasmin through the hydrolysis of a single Arginine-Valine bond. The catalytic site of t-PA is composed of amino acids His-322, Asp-371 and Ser-478. t-PA is a poor plasminogen activator in the absence of fibrin. The amino-terminal region is composed of several domains, which are homologous to other proteins. These distinct domains are involved in several functions of the enzyme, including binding to fibrin, fibrin-specific plasminogen activation, binding to endothelial cell receptors and rapid clearance in vivo. One such domain, comprising amino acid residues 50 to 87 (E domain) is homologous to Human Epidermal Growth Factor and seems to be involved in fibrin binding, fibrin affinity and in vivo clearance. The t-PA cDNA was cloned and subsequently expressed in Chinese hamster ovary (CHO) cells.
Tissue plasminogen activator (t-PA) is a component of the mammalian fibrinolytic system responsible for the specific activation of plasminogen associated with fibrin clots (i.e. it is capable of dissolving blood clots). Tissue plasminogen activator-mediated clot dissolution shows increased level of fibrinopeptide-A in plasma, which is a direct marker of clot-bound thrombin (Weitz, Leslie et al. 1998). A combination of tissue plasminogen activator and currently known anti-thrombin drugs such as heparin and hirudinis often used in medication but this anti-thrombin acts only on free thrombin, and the clot-bound thrombin is resistant towards heparin and other the inhibitors due to their low affinity. Besides, these drugs do not affect the indirect promoters of further thrombin generation (e.g. Factor V and Factor VIII). Hence, new chimeric proteins need to be designed which activate plasminogen along with inhibition of clot bound thrombin as well as the indirect promoters of thrombin, such as Factor V and Factor VIII.
Work on post-thrombolytic plasmin activity and consequent thrombin generation clearly suggests that there are potent factors which lead to the re-activation of the coagulation pathway even when the pathological clot has been cleared by a clot-buster drug. In addition, the generated thrombin is itself a potent coagulation pathway activator. It is remarkable that thrombin also performs an anti-coagulant function, which is an elegant example of the “self-limiting” control mechanisms of the hemostasis system such that there is no ‘run away’ coagulation throughout the vasculature. Free thrombin makes a 1:1 high-affinity, non-covalent complex with thrombomodulin, a cell surface protein (Kurosawa, Galvin et al. 1987). Once the thrombin-thrombomodulin complex is formed its substrate specificity is redirected from a pro-coagulant mode to an anti-coagulant one, whereby it activates the Protein C anticoagulant pathway. Thus, even though thrombin alone can activate protein C but once it complexes with thrombomodulin it accelerates protein C activation by nearly a 1000-fold (Esmon and Owen 1981; Owen and Esmon 1981)).
Mature thrombomodulin contains different domains responsible for different functions, namely thrombin inhibition and protein C activation which reside in the epidermal growth factor-like domains of this large protein. Epidermal growth factor-like (EGF) domains 5 and 6 are responsible mainly for thrombin affinity and EGF 4, 5 and 6 domains, activate protein C together (Kurosawa, Stearns et al. 1988; Stearns, Kurosawa et al. 1989). Thrombomodulin, or its isolated EGF domains 4, 5 and 6, are known to activate the protein C-centered anticoagulant pathway, and also directly inhibit thrombin's activity, but cannot dissolve the fibrin clots by themselves. For this, a thrombolytic agent is necessary. Both types of agents can, independently of each other, be used during cardiac thrombotic maladies, but the advantages of a single drug with both types of attributes (which has not been demonstrated so far) are obvious.
The term ‘hemostasis’ refers to the balance between anticoagulant and procoagulant activities in the blood/vascular system, wherein normally, the blood components, particularly platelets, do not interact abnormally with the blood vessels' inner lining. In case of injury or disease condition, platelets tend to adhere, and as a result, blood coagulation factors start to accumulate and get activated at the site of this injury, which, although a response to initiate repair at the site of injury, results in blood occlusion, thereby often precipitating thrombotic crises.
Blood coagulation and dissolution of a clot, inside blood vessels, are both necessary physiological processes for normal hemostasis. Clot formation and dissolution mechanisms inside the blood vessel are well known in the literature, and the role of different pro-coagulant proteins (clot promoting) and anti-coagulant proteins are now fairly also well-studied. Clot formation is the result of a complex set of reactions wherein thrombin plays a central role in initiation as well as coagulation cascade acceleration, with the end-result appearing in the form of a stable fibrin mesh.
Every modern thrombolytic/fibrinolytic therapy usually combines an anti-thrombotic medication as well (such as aspirin, heparin etc) which is required for the maintenance of normal equilibrium between the pro-coagulants and anti-coagulants during the clot lysis. During lysis, the transiently released thrombin initiates a self-generation loop to amplify clot growth; sometimes this results in a shift of the equilibrium towards the reformation of clot, or activation of the pro-coagulants/clot promoters.
Thrombolytics dissolve pathological clots by activating intrinsic plasminogen in the circulatory system. Among the available and therapeutically useful thrombolytics, streptokinase exhibits the highest thrombolytic potential but sometimes it leads to hemorrhage during the medication because of a lack of clot specificity. This problem is relatively less associated with other thrombolytics (tPA and SAK) because of their relatively increased fibrin clot specificity, but other shortcomings of these thrombolytics are lesser fibrinolytic potential and shorter in vivo half life. All the thrombolytics have essentially same mechanism of thrombolysis (plasminogen activation), but share a common problem, in that that after thrombolysis the generated thrombin often further amplifies thrombin generation by a feed-back mechanism controlled by blood coagulation factor Va and factor VIIIa. In the thrombolysis process, the clot-bound thrombin is also released into the circulation which is relatively resistant to in-built anti-thrombin and other externally provided thrombin inhibitory drugs. This clot-bound thrombin, present at the near vicinity of the ‘original’ injury also helps to generate even more thrombin in the vicinity of the clot/site of injury, particularly after incomplete removal of clot, which leads to the clinically grave, early re-occlusion problem.
Thombin generation during the lysis and activation of other intrinsic pro-coagulants is a normal part of the hemostasis equilibrium. Anti-thrombin agents like heparin, hirudin and chemically synthesized drugs can inhibit thrombin and prevent its subsequent transient effects, like suppression of hyper-responsive platelet formation process, inhibition of fibrinogen to fibrin conversion and other clot promoting activities which induces hyper-coagulibility in blood. Notably, however, all these direct inhibitors act on transiently generated thrombin, but not on those factors which actually speed up thrombin generation and play key roles in the procoagulant pathway. Thus, none of these drugs acts as efficaciously as desired on blood coagulation factors V and VIII, which play crucial role in thrombin generation and early re-occlusion.
The well-known cell surface molecule thrombomodulin, which makes 1:1 complexes with thrombin and directs its pro-coagulant function into a potent anti-coagulant, namely protein C activator. Both activated protein C and protein S degrade activated factor V and factor VIII (Esmon 1989).
Although all available thrombolytics do possess clot dissolving capability and their mechanism is well evident in literature, but during clot lysis, remnants of clot and plasmin lead to thrombin generation; at the same time, since these agents cannot inactivate the thrombin which makes a transient appearance during thelysis of the clot, the result is that reocclusion is a common problem subsequent to thrombolytic therapy. To date, no anti-thrombin drugs are available with a combination of thrombolytic and anti-thrombin properties, especially the pro-coagulant activity so as to inactivate the ‘real culprit’ which is chiefly responsible for re-thrombosis.